Transgenic animal models of Alzheimer's disease are described along with a method of using the transgenic animal models to screen for therapeutics useful for the treatment of Alzheimer's disease.
Alzheimer's disease (AD) is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia (The early story of Alzheimer's Disease, edited by Bick et al. (Raven Press, New York 1987)). It is the leading cause of dementia in elderly persons. AD patients have increased problems with memory loss and intellectual functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibit personality changes, socially inappropriate actions and schizophrenia (A Guide to the Understanding of Alzheimer's Disease and Related Disorders, edited by Jorm (New York University Press, New York 1987). AD is devastating for both victims and their families, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration.
The impact of AD on society and on the national economy is enormous. It is expected that the demented elderly population in the United States will increase by 41% by the year 2000. It is expensive for the health care systems that must provide institutional and ancillary care for the AD patients at an estimated annual cost of $40 billion (Jorm (1987); Fisher, “Alzheimer's Disease”, New York Times, Aug. 23, 1989, page D1, edited by Reisberg (Me Free Press, New York & London 1983)). These factors imply action must be taken to generate effective treatments for AD.
At a macroscopic level, the brains of AD patients are usually smaller, sometimes weighing less than 1,000 grams. At a microscopic level, the histopathological hallmarks of AD include neurofibrillary tangles (NFT), neuritic plaques, and degeneration of neurons. AD patients exhibit degeneration of nerve cells in the frontal and temporal cortex of the cerebral cortex, pyramidal neurons of hippocampus, neurons in the medial, medial central, and cortical nuclei of the amygdala, noradrenergic neurons in the locus coeruleus, and the neurons in the basal forebrain cholinergic system. Loss of neurons in the cholinergic system leads to a consistent deficit in cholinergic presynaptic markers in AD (Fisher (1983); Alzheimer's Disease and Related Disorders, Research and Development edited by Kelly (Charles C. Thomas, Springfield, Ill. 1984)). In fact, AD is defined by the neuropathology of the brain.
AD is associated with neuritic plaques measuring up to 200 μm in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the principal constituents of neuritic plaques is amyloid, which is stained by Congo Red (Fisher (1983); Kelly (1984)). Amyloid plaques stained by Congo Red are extracellular, pink or rust-colored in bright field, and birefringent in polarized light. The plaques are composed of polypeptide fibris and are often present around blood vessels, reducing blood supply to various neurons in the brain.
Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mechanisms of AD neuropathy. However, available evidence strongly indicates that there are distinct types of genetic predispositions for AD. First, molecular analysis has provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate et al. Nature 349:704–706 (1991); Murrell et al. Science 254:97–99 (1991); Chartier-Harlin et al. Nature 353:844–846 (1991); Mullan et al., Nature Genet. 1:345–347 (1992)). Additional genes for dominant forms of early onset AD reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature 376:775–778 (1995); Levy-Lahad et al., Science 269:973–977 (1995); Sherrington et al., Nature 375:754–760 (1995)). Another loci associated with AD resides on chromosome 19 and encodes a variant form of apolipoprotein E (Corder, Science 261:921–923 (1993).
Amyloid plaques are abundantly present in AD patients and in Down'Syndrome individuals surviving to the age of 40. The overexpression of APP in Down's Syndrome is recognized as a possible cause of the development of AD in Down's patients over thirty years of age (Rumble et al., New England J. Med. 320:1446–1452 (1989); Mann et al., Neurobiol. Aging 10:397–399 (1989)). The plaques are also present in the normal aging brain, although at a lower number. These plaques are made up primarily of the amyloid β peptide (Aβ; sometimes also referred to in the literature as β-amyloid peptide or β peptide) (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885–890 (1984)), which is also the primary protein constituent in cerebrovascular amyloid deposits. The amyloid is a filamentous material that is arranged in beta-pleated sheets. Aβ is a hydrophobic peptide comprising up to 43 amino acids. The determination of its amino acid sequence led to the cloning of the APP cDNA (Kang et al., Nature 325:733–735 (1987); Goldgaber et al., Science 235:877–880 (1987); Robakis et al., Proc. Natl. Acad. Sci. 84:4190–4194 (1987); Tanzi et al., Nature 331:528–530 (1988)) and genomic APP DNA (Lemaire et al., Nucl. Acids Res. 17:517–522 (1989); Yoshikai et al., Gene 87, 257–263 (1990)). A number of forms of APP cDNA have been identified, including the three most abundant forms, APP695, APP751, and APP770. These forms arise from a single precursor RNA by alternate splicing. The gene spans more hand 175 kb with 18 exons (Yoshikai et al. (1990)). APP contains an extracellular domain, a transmembrane region and a cytoplasmic domain. Aβ consists of up to 28 amino acids just outside the hydrophobic transmembrane domain and up to 15 residues of this transmembrane domain. Thus, Aβ is a cleavage product derived from APP which is normally found in brain and other tissues such as heart, kidney and spleen. However, Aβ deposits are usually found in abundance only in the brain.
The larger alternate forms of APP (APP751, APP770) consist of APP695 plus one or two additional domains. APP751 consists of all 695 amino acids of APP695 plus an additional 56 amino acids which has homology to the Kunitz family of serine protease inhibitors (KPI) (Tanzi et al. (1988); Weidemann et al., Cell 57:115–126 (1989); Kitaguchi et al., Nature 331:530–532 (1988); Tanzi et al., Nature 329:156 (1987)). APP770 contains all 751 amino acids of APP751 and an additional 19 amino acid domain homologous to the neuron cell surface antigen OX-2 (Weidemann et al. (1989); Kitaguchi et al. (1988)). Unless otherwise noted, the amino acid positions referred to herein are the positions as they appear in APP770. The amino acid number of equivalent positions in APP695 and APP751 differ in some cases due to the absence of the OX-2 and KPI domains. By convention, the amino acid positions of all forms of APP are referenced by the equivalent positions in the APP770 form. Unless otherwise noted, this convention is followed herein. Unless otherwise noted, all forms of APP and fragments of APP, including all forms of Aβ, referred to herein are based on the human APP amino acid sequence. APP is post-translationally modified by the removal of the leader sequence and by the addition of sulfate and sugar groups.
Van Broeckhaven et al., Science 248:1120–1122 (1990), have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This was confirmed by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al., Science 248:1124–1128 (1990)). The mutation substituted a glutamine for glutamic acid at position 22 of the Aβ (position 618 of APP695, or position 693 of APP770). In addition, certain families are genetically predisposed to Alzheimer's disease, a condition referred to as familial Alzheimer's disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full length protein (Goate et al. (1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the families and are absent in families with late-onset AD. This mutation at amino acid 717 increases the production of the Aβ1-42 form of Aβ from APP (Suzuki et al., Science 264:1336–1340 (1994)). Another mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al. (1992)). This mutation to amino acids 670 and 671 increases the production of total Aβ from APP (Citron et al., Nature 360:622–674 (1992)).
There are no robust animal models to study AD, although aging nonhuman primates seem to develop amyloid plaques of Aβ in brain parenchyma and in the walls of some meningeal and cortical vessels. Although aged primates and canines can serve as animal models, they are expensive to maintain, need lengthy study periods, and are quite variable in the extent of pathology that develops.
There are no spontaneous animal mutations with sufficient similarities to AD to be useful as experimental models. Various models have been proposed in which some AD-like symptoms may be induced by electrolysis, transplantation of AD brain samples, aluminum chloride, kainic acid or choline analogs (Kisner et al., Neurobiol. Aging 7:287–292 (1986); Mistry et al., J Med Chem 29:337–343 (1986)). Flood et al., Proc. Natl. Acad. Sci. 88:3363–3366 (1986), reported amnestic effects in mice of four synthetic peptides homologous to the Aβ. Because none of these share with AD either common symptoms, biochemistry or pathogenesis, they are not likely to yield much useful information on etiology or treatment.
Several transgenic rodent lines have been produced that express either the human APP gene or human APP complementary DNA regulated by a variety of promoters. Transgenic mice with the human APP promoter linked to E. coli β-galactosidase (Wirak et al., The EMBO J 10:289–296 (1991)) as well as transgenic mice expressing the human APP751 cDNA (Quon et al. Nature 352:239–241 (1991)) or subfragments of the cDNA including the Aβ (Wirak et al., Science 253:323–325 (1991); Sandhu et al., J. Biol. Chem. 266:21331–21334 (1991); Kawabata et al., Nature 354:476–478 (1991)) have been produced. Results obtained in the different studies appear to depend upon the source of promoter and the protein coding sequence used. For example, Wirak et al., Science 253:323–325 (1991), found that in transgenic mice expressing a form of the Aβ, intracellular accumulation of “amyloid-like” material, reactive with antibodies prepared against Aβ were observed but did not find other histopathological disease symptoms. The intracellular nature of the antibody-reactive material and the lack of other symptoms suggest that this particular transgenic animal is not a faithful model system for Alzheimer's disease. Later studies have shown that similar staining is seen in non-transgenic control mice and Wirak et al., Science 253:323–325 (1991) was partially retracted in a comment in Science 255:143–145 (1992). Thus, the staining seen by Wirak et al. appears to be artifactual.
Kawabata et al. (1991) report the production of amyloid plaques, neurofibrillary tangles, and neuronal cell death in their transgenic animals. In each of these studies, Aβ or a fragment containing Aβ was expressed. Wirak et al. (1991), used the human APP promoter while Kawabata et al. (1991) used the human thy-1 promoter. However, Kawabata et al. (1991) was later retracted by Kawabata et al., Nature 356:23 (1992) and Kawabata et al., Nature 356:265 (1992). In transgenic mice expressing the APP751 cDNA from the neuron-specific enolase promoter of Quon et al. (1991), rare, small extracellular deposits of material reactive with antibody prepared against synthetic Aβ were observed. A review of the papers describing these early transgenic mice indicate that do not produce characteristic Alzheimer pathologies (see Marx, Science 255:1200–1202 (1992)).
Transgenic mice expressing APP751 from a neuron-specific enolase (NSE) promoter were recently described by McConlogue et al., Neurobiol. Aging 15:S12 (1994), Higgins et al., Ann Neurol. 35:598–607 (1995), Mucke et al., Brain Res. 666:151–167 (1994), Higgins et al., Proc. Natl. Acad. Sci. USA 92:4402–4406 (1995), and U.S. Pat. No. 5,387,742 to Cordell. Higgins et al., Ann Neurol. 35:598–607 (1995) describe results with the same mice as described by Quon et al. (1991). Such mice have only sparse Aβ deposits which are more typical of very early AD and young Down's syndrome cases. The deposits seen in this transgenic mouse were also seen, although at a lower abundance, in non-transgenic control animals. Mature lesions such as frequent compacted plaques, neuritic dystrophy and extensive gliosis are not seen in these mice (Higgins et al., Ann Neurol. 35:598–607 (1995)). McConlogue et al. (1994) reported finding no Aβ deposits in these mice.
Transgenic mice in which APP is expressed from the neuronal specific synaptophysin promoter express APP at low levels equivalent to that in brain tissue from the NSE APP mice described above. These mice were also reported not to display any brain lesions (Higgins et al.).
Transgenic mice containing yeast artificial chromosome (YAC) APP constructs have also been made (Pearson and Choi, Proc. Natl. Acad. Sci. USA 90:10578–10582 (1993); Lamb et al., Nature Genetics 5:22–30 (1993); Buxbaum et al., Biochem. Biophys. Res. Comm. 197:639–645. (1993)). These mice contain the entire human APP genomic gene and express human APP protein at levels similar to endogenous APP; higher levels of expression than that obtained in mice using the NSE promoter. None of these mice, however, show evidence of pathology similar to AD.
Alzheimer's disease animal models, including transgenic models, have been recently reviewed by Lannfelt et al., Behavioural Brain Res. 57:207–213 (1993), and Fukuchi et al., Ann. N.Y. Acad. Sci. 695:217–223 (1993). Lannfelt et al. points out that none of the prior transgenic animals that show apparent plaques demonstrate neuropathological changes characteristic of AD. Lannfelt et al. also discusses possible reasons for the “failure” of previous transgenic animal models. Similarly, Fukuchi et al. discusses the failure of prior transgenic animal models to display most of the characteristics known to be associated with AD. For example, the transgenic mouse reported by Quon et al. is reported to produce Aβ immunoreactive deposits that stain only infrequently with thioflavin S and not at all with Congo Red, in contrast to the staining pattern of AD Aβ deposits.
Alzheimer's disease is characterized by numerous changes in the expression levels of various proteins, the biochemical activity and histopathology of brain tissue, as well as cognitive changes in affected individuals. Such characteristic changes associated with AD have been well documented. The most prominent change, as noted above, is the deposition of Aβ into amyloid plaques (Haass and Selkoe, Cell 75:1039–1042 (1993)). A variety of other molecules are also present in plaques, such as apolipoprotein E, laminin, amyloid P component, and collagen type IV (Kalaria and Perry, Brain Research 631:151–155 (1993); Ueda et al., Proc. Natl. Aca. Sci. USA 90:11282–11286 (1993)). Changes in cytoskeletal markers have also been associated with AD, such as the changes in microtubule-associated protein tau, MAP-2 or neurofilaments (Kosik et al., Science 256: 780–783 (1992); Lovestone and Anderton, Current Opinion in Neurology & Neurosurgery 5:883–888 (1992); Brandan and Inestrosa, General Pharmacology 24:1063–1068 (1993); Trojanowski et al., Brain Pathology 3:45–54 (1993); Masliah et al., American Journal of Pathology 142:871–882 (1993)). Alzheimer's disease is also known to stimulate an immunoinflammatory response, increasing such inflammatory markers as glial fibrillary acidic protein (GFAP), α2-macroglobulin, and interleukins 1 and 6 (IL-1 and IL-6) (Frederickson and Brunden, Alzheimer Disease and Associated Disorders 8:159–165 (1994); McGeer et al., Canadian Journal of Neurological Sciences 18:376–379 (1991); Wood et al., Brain Research 629:245–252 (1993)). Finally, neuronal and neurotransmitter changes have been associated with AD, such as the cholinergic, muscarinic, serotinergic, adrenergic, and adensosine receptor systems (Rylett et al., Brain Res 289:169–175 (1983); Sims et al., Lancet 1:333–336 (1980); Nitsch et al., Science 258:304–307 (1992); Masliah and Terry, Clinical Neuroscience 1:192–198 (1993); Greenamyre and Maragos, Cerebrovascular and Brain Metabolism Reviews 5:61–94 (1993); McDonald and Nemeroff, Psychiatric Clinics of North America 14:421–422 (1991); Mohr et al., Journal of Psychiatry & Neuroscience 19:17–23 (1994)).
It is therefore an object of the present invention to provide an animal model for Alzheimer's disease that is constructed using transgenic technology.
It is a further object of the present invention to provide transgenic animals characterized by certain genetic abnormalities in the expression of the amyloid precursor protein.
It is a further object of the present invention to provide transgenic animals exhibiting one or more histopathologies similar to those of Alzheimer's disease.
It is a further object of the present invention to provide transgenic animals expressing one or more Aβ-containing proteins at high levels in brain tissue.
It is a further object of the present invention to provide a method of screening potential drugs for the treatment of Alzheimer's disease using transgenic animal models.